A kinetic trapping approach for facile access to 3FaxNeu5Ac and a photo-crosslinkable sialyltransferase probe

Sialic acid (Neu5Ac) is installed onto glycoconjugates by sialyltransferases (STs) using cytidine monophosphate-Neu5Ac (CMP-B-D-Neu5Ac) as their donor. The only class of cell-active ST inhibitors are those based on a 3FaxNeu5Ac scaffold, which is metabolically converted into CMP-3FaxNeu5Ac within cells. It is essential for the fluorine to be axial, yet stereoselective installation of fluorine in this specific orientation is challenging. Sialic acid aldolase can convert 3-fluoropyruvate and 2-acetamido-2-deoxy-D-mannopyranose (ManNAc) to 3FNeu5Ac but stereocontrol of the fluorine in the product has not been possible. We hypothesized that the 3Fax kinetic product of a sialic acid aldolase reaction could be trapped by coupling with CMP-sialic acid synthetase, to yield CMP-3FaxNeu5Ac. Here, we report that highly active CMP-sialic acid synthetase and short reaction times produces exclusively CMP-3FaxNeu5Ac. Removal of CMP from CMP-3FaxNeu5Ac, under acidic conditions unexpectedly led to 3-fluoro-B-D-Neu5Ac 2-phosphate (3FaxNeu5Ac-2P). Alkaline phosphatase enabled the successful conversion of 3FaxNeu5Ac-2P to 3FaxNeu5Ac, enabling the stereochemically-controlled access to 3FaxNeu5Ac, which is effective in lowering the sialoglycan ligands for Siglecs on cells. Moreover, our kinetic trapping approach could be used to access CMP-3FaxNeu5Ac with modifications at the C5, C9, or both positions, which enabled the chemoenzymatic synthesis of a photo-crosslinkable version of CMP-3FaxNeu5Ac that selectively photo-crosslinked to ST6Gal1 over two other sialyltransferases.